Buffer solutions having selective bactericidal activity against gram negative bacteria and methods of using same

ABSTRACT

Buffer solutions for pharmaceutical preparations that have bactericidal activity preferentially against gram negative bacteria are provided. The buffers have a pH of greater than about 10 or less than about 4.5 with low buffer capacity. Methods of their use in reducing the occurrence of blood stream infections in a mammal is also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of U.S. patent application Ser. No.13/022,005, filed Feb. 7, 2011, which is a Continuation of U.S. patentapplication Ser. No. 12/276,707, filed Nov. 24, 2008, which is aContinuation of U.S. patent application Ser. No. 12/205,200, filed Sep.5, 2008, which claims priority to U.S. Patent Application 60/970,716,filed Sep. 7, 2008, the entire contents of which are incorporated hereinby reference.

FIELD OF THE INVENTION

The present invention relates generally to the field of buffer solutionshaving bacteriostatic and/or bactericidal activity. More specifically,the present invention relates to buffer solutions that have bactericidalactivity preferentially against gram negative bacteria.

BACKGROUND OF THE INVENTION

The use of buffers to maintain a pH and solubilize or dilute activepharmaceutical agents (“APIs”) before administration (e.g., byinjection) is routine. Many buffers, however, contain components thatmaintain a neutral pH and foster microbial growth, which can lead tosepsis and other undesirable infection-related complications.

Gram negative bacteria are a particularly troublesome class of microbes,as they are commonplace in the hospital environments and difficult toeradicate and/or control. Infections with this class of bacteria tend tohave higher morbidity/mortality rates when a patient becomes septic, inpart, because gram negative bacteria are especially difficult organismsto treat. Also, gram negative bacteria are associated with watercontamination which can occur with chronic indwelling catheters such asused with intravenous administration. Hence, there is a need for buffersystems that have anticidal activity with specificity to gram negativebacteria.

SUMMARY OF THE INVENTION

In one embodiment of the present invention, a method of selectivelykilling gram negative bacteria and inhibiting the growth of grampositive bacteria in a pharmaceutical preparation comprising an activeagent is provided, the method comprising supplying the active agent witha buffer having a pH of greater than about 10 or less than about 4.5 anda low buffer capacity, wherein the pharmaceutical preparation does notcomprise epoprostenol sodium as the sole active agent. In addition tobacteria, the buffer may further inhibit the growth of fungus, mold, orboth. Preferably, the buffer has a pH between about 10 to about 12, morepreferably a pH between about 10.2 to about 10.8. In other embodiments,the buffer has a pH between about 3 and 4.5, more preferably a pHbetween about 3.5 and 4.5.

The buffer may comprise glycine; and in a specific embodiment, thebuffer is sterile diluent for FLOLAN®, namely a buffer comprisingglycine and sodium hydroxide, added to adjust the pH to 10.2 to 10.8.The active agent may be any active pharmaceutical agent that requiressolution or dilution with a buffer and may be injected (e.g.,intravenously). The active agent may be treprostinil sodium (sometimesreferred to herein as treprostinil), preferably supplied at aconcentration between about 0.004 mg/mL to about 0.13 mg/mL treprostinilsodium.

The buffer may comprise sorbic acid or citric acid or any other weakacid that is pharmaceutically acceptable for parenteral use. The pH canbe adjusted with hydrochloric acid or sodium hydroxide to attain a finalpH between 3 and 4.5. The active agent may be any active pharmaceuticalagent that requires solution or dilution with a buffer and may beinjected (e.g., intravenously).

In another embodiment of the invention, a method of reducing theoccurrence of blood stream infections in a mammal being treated with anactive agent is provided, the method comprising administering to themammal the active agent with a buffer having a pH of greater than about10 or less than about 4.5 and a low buffer capacity, wherein the activeagent is not epoprostenol sodium, and wherein the administration reducesthe gram negative bacteria and inhibits the growth of gram positivebacteria. In some cases, the human subject may suffer from pulmonaryarterial hypertension.

Preferably, the buffer has a pH between about 10 to about 12, morepreferably a pH between about 10.2 to about 10.8 and a low buffercapacity. Alternatively, the buffer has a pH preferably between about 3to about 4.5, more preferably a pH between about 3.5 to about 4.5 and alow buffer capacity. The buffer may comprise glycine; and in a specificembodiment, the buffer is sterile diluent for FLOLAN®. The active agentmay be any active pharmaceutical agent that requires solution ordilution with a buffer and may be injected (e.g., intravenously). Theactive agent may be treprostinil sodium, preferably supplied at aconcentration between about 0.004 mg/mL to about 0.13 mg/mLtreprostinil. Selection of the buffer will depend on the desired pH.While the buffer components should have a pKa close to the desired pH,the buffer capacity should be low to avoid pH changes in the blood uponinfusion. A preferred buffer capacity for such buffers is 0.01 and less.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a chromatogram of a “blank” injection of BWFI (A) andtreprostinil diluted with BWFI (B).

FIG. 2 is a chromatogram of a “blank” injection of BNS (A) andtreprostinil diluted with BNS (B).

FIG. 3 is a chromatogram of Sterile Diluent for FLOLAN® (A) andtreprostinil diluted with same (B).

FIG. 4 is a chromatogram of 0.004 mg/mL treprostinil in Sterile Diluentfor FLOLAN® at T₀ (A) and T_(initial) (B).

FIG. 5 is a chromatogram of 0.13 mg/mL treprostinil in Sterile Diluentfor FLOLAN® at T₀ (A) and T_(initial) (B).

FIG. 6 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Staphylococcus aureus, a grampositive bacterium, in a pharmaceutical preparation comprising 0.004mg/mL. Values for ≦Log 1 (treprostinil in sterile diluent) or ≧Log 6.48(treprostinil in WFI, NS) are recorded as Log 1 and Log 6.48,respectively.

The legend for FIGS. 6-15 is as follows:

(open circles): FLOLAN® in Sterile Diluent for FLOLAN®

(closed circles): Treprostinil in Sterile Diluent for FLOLAN®

(open squares): Treprostinil in sterile water for injection

(closed squares): Treprostinil in bacteriostatic water for injection

(open diamonds): Treprostinil in normal (0.9%) saline

(closed diamonds): Treprostinil in bacteriostatic normal saline

(open triangle): Treprostinil in 5% dextrose in water for injection(D5W).

FIG. 7 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Escherichia coli, a gramnegative bacterium, in a pharmaceutical preparation comprising 0.004mg/mL treprostinil. Values for ≦Log 1 (treprostinil in sterile diluent)recorded as Log 1.

FIG. 8 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Pseudomonas aeruginosa, a gramnegative bacterium, in a pharmaceutical preparation comprising 0.004mg/mL treprostinil. Values≦Log 1 (treprostinil in sterile diluent) or≧6.48 (treprostinil in D5W) are recorded as Log 1 and Log 6.48,respectively

FIG. 9 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Candida albicans, a fungus, ina pharmaceutical preparation comprising 0.004 mg/mL treprostinil.

FIG. 10 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Aspergillus niger, a mold, in apharmaceutical preparation comprising 0.004 mg/mL treprostinil.

FIG. 11 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Staphylococcus aureus, a grampositive bacterium, in a pharmaceutical preparation comprising 0.13mg/mL treprostinil. Values for ≦Log 1 (treprostinil in sterile diluent)or ≧Log 6.48 (treprostinil in WFI, NS, D5W) are recorded as Log 1 andLog 6.48, respectively.

FIG. 12 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Escherichia coli, a gramnegative bacterium, in a pharmaceutical preparation comprising 0.13mg/mL treprostinil. Values for ≦Log 1 (treprostinil in sterile diluent)or ≧Log 6.48 (treprostinil in NS) are recorded as Log 1 and Log 6.48,respectively.

FIG. 13 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Pseudomonas aeruginosa, a gramnegative bacterium, in a pharmaceutical preparation comprising 0.13mg/mL treprostinil. Values for ≦Log 1 (treprostinil in sterile diluent)recorded as Log 1.

FIG. 14 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Candida albicans, a fungus, ina pharmaceutical preparation comprising 0.13 mg/mL treprostinil.Value≧Log 3.48 at time 0.25 hours for treprostinil in NS recorded as Log3.48.

FIG. 15 is a graph showing the antimicrobial activity (CFU) over time(days) of various buffer systems against Aspergillus niger, a mold, in apharmaceutical preparation comprising 0.13 mg/mL treprostinil.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention is directed to the use of buffer systems tomaintain a specific pH range as anticidal agents in pharmaceuticalpreparations. The term “buffer” as used herein refers to any solutionwith a controlled pH that may serve to dissolve a solid (e.g.,lyophilized) pharmaceutical or as a diluent to dilute a liquidpharmaceutical. According to the invention, the buffers described hereinmaintain a pH that exhibits bacteriostatic activity toward most, if notall, microbes, including bacteria, molds and fungi and further exhibitbactericidal activity toward gram negative bacteria. Examples of gramnegative bacteria include Escherichia coli, Pseudomonas aeruginosa,Salmonella, Moraxella, Helicobacter, Stenotrophomonas, Bdellovibrio,Legionella, Neisseria gonorrhoeae, and Neisseria meningitidis. Gramnegative bacteria are a common source of infection in hospitalenvironments and therefore buffers that maintain a pH above 10 or lessthan about 4.5 with low buffer capacity have bactericidal activityspecific for gram negative bacteria are desirable. By way of example,gram positive bacteria include Staphylococcus aureus Bacillus, Listeria,Staphylococcus, Streptococcus, Enterococcus, and Clostridium.

“Bacteriostatic” is defined as the ability to retard or prevent theexpansion of a microbe that might be present, or become present, in thebuffer solution. In other words, “bacteriostatic” activity does notinclude bactericidal activity, which is defined herein as activity thatkills a microbe that might be present, or become present, in the buffer.Microbes are broadly defined herein to include unicellular organisms,such as, for example, bacteria, molds, and fungi.

The present inventors have learned that buffers having high pH (>10) orlow pH (<4.5) have bactericidal activity specific for gram negativebacteria and bacteriostatic activity toward gram positive bacteria andother microbes. Without being held to or bound by theory, it iscurrently believed that differences in the biochemistry, perhaps cellwall biochemistry, between gram negative and gram positive bacteria mayaccount for their differential sensitivity toward high pH buffers. Inthe context of the present invention, “high” pH is a pH value of about 9to about 12, preferably about 10 to about 12. In a preferred embodimentof the invention, buffers have a pH of about 10.2 to about 10.8 or about3.5 to about 4.5.

In addition to high pH, the present inventors have learned the bufferscomprising glycine are particularly advantageous. In such embodiments,glycine is present at a concentration (w/w) of about 30% to about 80%,preferably about 45% to about 65%, and most preferably, about 50% toabout 60%. The term “about” is used herein in recognition of theinherent inaccuracies in calculations and measurements in the art and toinclude nominal and accepted variations “about” the recited numeral.

In addition to glycine, buffers as described herein may comprise anyother buffer system, including those known in the art, that can maintaina pH in the ranges stated herein.

In a specific embodiment of the present invention, the diluent forFLOLAN® (epoprostenol sodium) employs glycine as a buffer component. Aswill be described in greater detail below, the diluent for FLOLAN® wasunexpectedly discovered to have specific anticidal activity toward gramnegative bacteria and bacteriostatic activity toward remainder microbes.The diluent for FLOLAN® comprises 50 mL of 94 mg glycine, 73.3 mg sodiumchloride, and sodium hydroxide, added to adjust the pH to 10.2 to 10.8.(About 44% NaCl in glycine.)

The buffers as described herein may be suitable for any activepharmaceutical ingredient (“API”) that is stable at high pH and providedthat the chemical properties of the API do not substantially drop the pHof the buffer below, for example, about 10. Hence, the followingexamples notwithstanding, the present invention should not be limited toany one or any one class of API nor, for that matter, a limited range ofconcentrations. Further, the novel and unexpected anticidal propertiesof the buffers may be especially suited for medicaments that areadministered by injection. Indeed, in one embodiment of the invention,it is anticipated that use of the high and low pH buffers as describedherein can reduce the occurrence of blood stream infections in a mammalbeing treated with an active agent. It should be noted, however, thatthe present invention is not limited to medicaments that are prescribedfor injection (including intravenous injection), but any medicament thatrequires solution and/or dilution (e.g., for oral administration).

In a specific embodiment of the present invention, the buffer systemsdescribed are used with treprostinil sodium. More specifically, as willbe next disclosed by way of examples, the diluent for FLOLAN® is used tobuffer treprostinil sodium.

EXAMPLES

A compatibility study of treprostinil with a 100-mL CADD delivery devicewas performed. More specifically, the compatibility and stability oftreprostinil diluted with bacteriostatic water for injection (“BWFI”) orbacteriostatic normal saline (“BNS”), both of which are preserved withparabens, was determined. The sample solutions were prepared at 0.004mg/mL and 0.13 mg/mL treprostinil, which comprises the entire range ofconcentrations at which treprostinil might be prescribed, and placed ina SIMS Deltec, Inc. CADD-Legacy™ 1 (Model 6400) Pump delivery devicethat was pumped continuously over a period of 52 hours while stored at40° C. and ambient relative humidity (“RH”).

At specified time points (e.g., T₀, initial, 24 hours, and 52 hours),samples were collected from the distal end of the tube after pump andcharacterized for appearance, pH, and concentration of treprostinil.Furthermore, the solutions were subjected to antimicrobial effectivenesstesting (“AET”) over a similar time period of about 2 days. A similarexperimental procedure was followed for Flolan reconstituted solutions.However, sterility and AET testing were performed on FLOLAN® after only8 hours at room temperature on account of the medicament's limitedstability in solution.

The stability of treprostinil was monitored by a fully validatedstability indicating HPLC assay. In order to ascertain whetherparabens—present in the “bacteriostatic” solutions—would causeinterference in the chromatography with treprostinil, a preliminaryexperiment confirmed that the paraben “peaks” did not interfere with thetreprostinil “peak” Or any impurity “peak.” Solutions of BWFI and BNS,and treprostinil diluted in BWFI and BNS were analyzed using HPLC. FIGS.1 and 2 shows that the paraben peaks from either methyl- orethyl-paraben did not interfere (e.g., overlap) with the peak fortreprostinil. There was also no chromatographic interference oftreprostinil with Sterile Diluent for FLOLAN® (FIG. 3).

A low-level linearity study was also performed to cover the expectedconcentration range of treprostinil in the dilute solutions. Fivesolutions of treprostinil were prepared at 0.002, 0.01, 0.05, 0.1, and0.15 mg/mL (diluted from the 1.0 mg/mL standard solution) and eachsolution was injected in duplicate. The intention was to prove linearitybetween the detector response and treprostinil concentration within thediluted concentration range in order to use a single-point standard at0.1 mg/mL during the analysis. The detector response for treprostinilwas determined to be linear from 0.002 to 0.15 mg/mL. The correlationcoefficient (r) for the experiment was 0.999995, meeting the requirementof at least 0.999.

Solutions of 0.004 mg/mL treprostinil in BWFI, BNS, or Sterile Diluentfor FLOLAN® were prepared from the 1.0 mg/mL strength of Remodulin.Solutions of 0.13 mg/mL treprostinil in BWFI and in BNS were preparedfrom the 10 mg/mL strength of Remodulin. Vials of FLOLAN® werereconstituted with 5 mL of Sterile Diluent for FLOLAN® using theprocedure outlined in the package insert.

A portion (approximately 2 mL) of each of the four solutions was removedfor T₀ analysis. The remaining solution was loaded into each of fourseparate SIMS Deltec, Inc. 100-mL Medication Cassette™ Reservoirs. Thecassettes and tubing were attached to the CADD-Legacy™ 1 Pump followingthe manufacturers instructions. The four cassette/CADD pump sets wereplaced in a 40° C./Ambient RH chamber. A needle at the end of the tubingwas placed into a sealed HPLC vial (with needle vent). The flow on thepump was set to 40 mL/24 hours and started. The solution from each pumpwas collected into separate HPLC vials (for about an hour) for testingat the “Initial” interval. The needle was then transferred to a sealedwaste container (with needle vent). At 24 and 52 hours, the solution wascollected again into a new, sealed HPLC vial for testing.

The solutions collected at T₀, initial, 24 hours, and 52 hours wereanalyzed for physical appearance, pH, and assayed by HPLC fortreprostinil. Tables 1 and 2 summarize the results for treprostinildiluted with BWFI and BNS, respectively. The appearance of all solutionswas clear and colorless, free from visible particulate matter. Hence,the results show no compatibility problems for the treprostinil solutionin BWFI or BNS at either concentration.

TABLE 1 Chemical testing results for treprostinil in BWFI TestingInterval Concentration 24 52 (mg/mL) Testing Preparation T₀ T_(initital)hours hours 0.004 Treprostinil 1 104.1 99.5 100.8 99.6 Assay 2 100.6100.6 101.1 101.0 (% LC) Average 102.4 100.0 101.0 100.3 pH NA 6.7 6.86.8 6.8 0.13 Treprostinil 1 100.1 99.6 100.4 101.2 Assay 2 100.0 99.8100.5 100.9 (% LC) Average 100.0 99.7 100.5 101.0 pH NA 6.7 6.9 6.8 7.0LC: Label claim

TABLE 2 Chemical testing results for treprostinil in BNS TestingInterval Concentration 24 52 (mg/mL) Testing Preparation T₀ T_(initital)hours hours 0.004 Treprostinil 1 97.8 94.8 96.7 99.9 Assay 2 102.9 94.097.9 102.3 (% LC) Average 100.4 94.4 97.3 101.1 pH NA 6.4 6.6 6.6 6.60.13 Treprostinil 1 100.1 96.3 99.8 100.3 Assay 2 100.5 96.0 99.7 100.2(% LC) Average 100.3 96.1 99.7 100.2 pH NA 6.3 6.7 6.7 6.5 LC: Labelclaim

Similar results were obtained for the solutions of treprostinil inSterile Diluent for FLOLAN®, which are summarized in Table 3. Theappearance of all solutions was clear, colorless and free from visibleparticulate matter. The results also show no compatibility problems forthe treprostinil solutions in Sterile Diluent for FLOLAN® fortreprostinil at either concentration (FIGS. 4 and 5). Hence, the resultsshow no compatibility problems for the dilute treprostinil solutions inany of the diluent solutions at either concentration.

TABLE 3 Chemical testing results for treprostinil in Sterile Diluent forFLOLAN ® Testing Interval Concentration 24 52 (mg/mL) TestingPreparation T₀ T_(initital) hours hours 0.004 Treprostinil 1 95.9 108.4100.6 100.7 Assay 2 96.1 108.9 101.4 101.2 (% LC) Average 96.0 108.7101.0 101.0 pH NA 10.6 10.5 10.6 10.5 0.13 Treprostinil 1 100.3 102.9101.4 102.3 Assay 2 100.2 102.7 101.4 102.1 (% LC) Average 100.2 102.8101.4 102.2 pH NA 10.5 10.4 10.5 10.5 LC: Label claim

For the treprostinil solutions, after 52 hours in the cassette at 40°C./Ambient RH, the solutions were removed and AET was performedaccording to USP NF 24 Supplement 2<51> with an inclusion of a 48 hourplating for all organisms. For the FLOLAN® solution, the testing wasperformed following the same procedure, but after the solution had beenin the cassette for 8 hours at room temperature. FLOLAN® was also testedfor sterility.

The AET USP requirements for a Category 1 product, which includesparenteral solutions, are as follows: for bacteria, there must not beless than a 1.0 log reduction from the initial calculated count at 7days and not less than a 3.0 log reduction from the initial count at 14days and no increase from the 14 days' count at 28 days. For the yeastand mold, there should be no increase from the initial calculated countat 7, 14 and 28 days.

While FLOLAN® diluted in sterile diluent for Flolan met the USPrequirements for AET, the treprostinil solutions in BWFI and BNS failed.These dilute treprostinil solutions failed AET because the bacterialreduction rate was not sufficient, mainly for gram negative bacteria.However, treprostinil in Sterile Diluent for FLOLAN® met the USPcriteria. See FIGS. 6 to 15.

While the invention has been described in connection with specificembodiments thereof, it will be understood that it is capable of furthermodifications and this application is intended to cover any variations,uses, or alterations of the invention following. In general, theprinciples of the invention and including such departures from thepresent disclosure as come within known or customary practice within theart to which the invention pertains and as may be applied to theessential features hereinbefore set forth and as follows in the scope ofthe appended claims.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those of ordinary skill in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

What is claimed is:
 1. A method of reducing occurrence of a bacterialinfection in a human suffering from pulmonary arterial hypertension, whois undergoing treatment for said pulmonary hypertension, associated withoccurrence of a bacterial infection comprising diluting a startingsolution of an active pharmaceutical ingredient other than epoprostenolwith a buffer comprising glycine and having a pH of greater than 10 toprovide a final solution with a pH of greater than 10 and an amount ofthe active pharmaceutical ingredient other than epoprostenol effectivefor treating pulmonary arterial hypertension, and administering saidfinal solution to the human subject in need thereof.
 2. The method ofclaim 1, wherein the buffer further comprises sodium hydroxide.
 3. Themethod of claim 1, wherein the buffer has a pH between 10 and
 12. 4. Themethod of claim 3, wherein the buffer has a pH between 10.2 and 10.8. 5.The method of claim 1, wherein the final solution is administered at aconcentration between about 0.001 mg/mL to about 1 mg/mL.
 6. The methodof claim 1, wherein the final solution is administered at aconcentration between about 0.004 mg/mL to about 0.13 mg/mL.
 7. Themethod of claim 1, wherein the administering is by injection.
 8. Themethod of claim 7, wherein the injection is intravenous injection. 9.The method according to claim 1, wherein the administration reduces thegrowth of gram negative bacteria.
 10. The method of claim 4, wherein thefinal solution is administered intravenously.
 11. The method of claim 1,wherein the buffer is a 50 mL solution of 94 mg of glycine, 73.3 mg ofsodium chloride, and sodium hydroxide.
 12. The method of claim 11,wherein the administering is by injection.
 13. The method of claim 12,wherein the injection is intravenous injection.